Microcapsule degradation

We implemented microencapsulation of engineered human cells and designed the system for degradation of alginate microcapsules.

Engineered HEK293T cells were succesfully incorporated into alginate microcapsules and extended time viability of the encapsulated cells was demonstrated.

Secretory alginate lyases from Pseudomoalteromonas elyakovii and from Pseudomonas aeruginosa were cloned by replacing the signal peptide of the alginate lyase with the eukaryotic signal peptide.

Alginate lyases were succesfully produced and secreted from HEK293T cells.

Degradation of alginate beads was demonstrated by alginate lyase from Sphingobacterium multivorum.

Degradation of alginate microcapsules

Our biopharmaceutical delivery system is based on microencapsulated mammalian cells which produce the required therapeutics. These cells are safely sealed in the alginate microcapsules, forming an immune-privileged environment for the therapeutic cells, and implanted into the tissue such as e.g. the eye or placenta. The semi-permeable capsule allows free transport of nutrients, signalling molecules, and produced protein therapeutics, while it prevents immune cells from reaching and destroying the implanted therapeutic cells. Therapeutic cells therefore do not need to be immuno-compatible for each individual patient and can threfore be optimized and mass produced to increase their efficiency and affordability of this therapy.

We designed our device to leave no trace after the therapy has been completed, by initiating secretion of an alginate-degrading enzyme to break down the microcapsules followed by the apoptosis of therapeutic cells. This approach should increase the safety and decrease the unwanted effects of treatment and makes the surgical removal of microcapsules or fibrotic tissue around microcapsules obsolete.

Figure 1. Schematic representation of alginate microcapsule and its degradation. Microcapsules serve as a semi-permeable membrane, allowing exchange of therapeutic and inducer molecules as well as nutrients and metabolites between encapsulated cells and the environment. On the other hand the microcapsule prevents the immune cells and immunoglobulin complexes to access the engineered cells while at the same time preventing uncontrolled dissemination of therapeutic cells throughout the body.

Alginate microcapsules

Alginate is the most widely used and clinicaly tested biomaterial for cell encapsulation. It is found in cell walls of brown seaweed. This polysaccharide is made of (1, 4)-linked monomeric units of β-D-mannuronate (M) and α-L-guluronate (G) (Figure 2). Consecutive M or G residues form so called M or G-blocks, whereas the alternating M and G units constitute MG-blocks of alginate (Duan et al., 2009).

Figure 2. Structure of alginate polymer. α-L-guluronate sugar residues is shown on the left and β-D-mannuronate on the right side. Source: http://en.wikipedia.org/wiki/Alginate

The host immune system imposes a great threat on our therapeutic cells as the human defence system could quickly locate and destroy our drug-producing cells since they lack the signatures of the endogenous cells. Alginate microcapsules are an almost ideal solution for this problem: the encapsulated cells are protected against the cytotoxic cells while the material used is biocompatible and allows exchange of smaller molecules, including proteins (Figure 1). It has been demonstrated that encapsulated cells remain viable for several months, therefore many therapeutic programs can be accomplished within this time frame. This sequestration technology also prevents the uncontrolled dissemination of our device throughout the patient's body (Ausländer et al., 2011).

Cell encapsulation is technically achieved by dropping a mixture of cells and liquid alginate into a calcium chloride solution which solidifies the droplets, transforming them into the hydrogel beads whose size in our system can be adjusted within the range of 300-500 μm in diameter. These beads are then coated with a polycation such as poly(L-lysine) (PLL) to form a membrane which further decreases the porosity of the wall of capsules. In the next step of cell encapsulation, an outer alginate layer is applied. This alginate coat also minimizes the attachment of patient's cells to the microcapsules due to the net negative charge of its surface (Vos et al., 2006). To allow cell growth and division within the alginate beads, the core of microcapsules is depolymerized with sodium citrate.

Figure 3. Büchi Encapsulator B-395 Pro was used for cell encapsulation.

Encapsulation and degradation of microcapsules for therapy

Hopefully, after a few weeks or months of treatment, the patient would recover and we could terminate the genetically engineered microencapsulated cells by inducing apoptosis. However, before their programmed death, production and secretion of the alginate-degrading enzyme would be induced. Alginate lyase can break down the alginate polymer of microcapsules, ideally leaving no trace of our biopharmaceutical delivery system.

Figure 4. Pseudomoalteromonas elyakovii alginate lyase structure model prepared by I-TASSER server (Roy et al., 2011). Chitin binding like-domain is depicted in brown and alginate lyase domain in blue. The enzyme was engineered for secretion from HEK293T cells by the addition of preprotrypsin sequence to the N-terminus. Additionally, protein without chitin binding like-domain was also prepared (mature alginate lyase).

Alginate lyases are not present in the mammalian genome but have been found in bacteria, algae and marine mollusks. Alginate lyase is able to depolymerise the alginate crosslinked by calcium (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others like G-rich more. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found from the literature the alginate lyase from bacterium Pseudoalteromonas elyakovii to be a promising candidate, since it can degrade all types of alginate (Sawabe et al., 2007). Since we could not obtain the gene for the described enzyme from the authors we decided to design a synthetic mammalian codon usage-optimized and BioBrick standard-compatible version of this bacterial gene for our iGEM project. Furthermore, because the selected alginate lyase consists of two domains, a putative chitin binding like-domain which is in bacterial expression system posttranslationally cleaved off and an alginate lyase domain representing the mature enzyme (Sawabe et al., 2007) (Figure 4), we also prepared a truncated version of P. elyakovii alginate lyase, abbreviated by chitin binding like-domain. We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of the secreted enzyme (Figure 5). The second implemented alginate lyase was from Pseudomonas aeruginosa strain PAO1 (bacteria were kindly provided by dr. Guillermo Martinez de Tejada from Univ. Navarra, Spain). This Gram-negative bacteria secretes alginate lyase to degrade the alginate that forms the mucoid matrix composing the bacterial biofilm (Schiller et al., 1993). This alginate lyase might therefore also be used for medical applications to degrade the viscous polysaccharide coat of Pseudomonas in patients with cystic fibrosis.

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